Generation of long-term human

pancreatic islet cell activity in vivo by

induction of immune tolerance to

human stem cells

John S. Pixley, M.D.

University of Nevada SOM, USA

Science, Vol. 102:400-401 (1945)

Immunogenetic Consequences of Vascular

Anastomoses between Bovine Twins

• Intermingling of siblings cells in placental

circulation results in long-term chimerism

• Immune tolerance is acquired during

development

• Existence of stem cells and their engraftment

The inoculum from A-line male suspension in ringer’s solution of small

organized tissue clumps, isolated cells and cell debris prepare by

prolonged chopping with scissors of testis, kidney and splenic tissue

0.01 ml injected via intraperitoneal injection into day 15-16 fetuses of a

CBA X CBA mating

After birth skin graft acceptance was determined

Summary

• Cellular inoculum during development results in

skin graft tolerance in adult life

• The tolerance is donor specific (i.e. full ability to

respond to 3rd party donor)

Intrauterine transplantation

IP injection on day

58-65

IP injection on

day 14-16

SHEEP MOUSE

Engraftment and Long-term Expression of Human

Fetal Hematopoietic Stem Cells in Sheep

Following Transplantation in Utero

Fetal age at transplantation day 48-54 term gestation 145 days

J Clin Inv 1992; 89: 1178-88

Prolonged hematopoietic chimerism in

normal mice transplanted in utero with

human hematopoietic stem cells

Pathobiology 1998; 66: 230-39

Peripheral blood

Thymus

Hu

ma

n C

D4

5

Side scatter

Engraftment receptivity is

gestational age dependent

Fetal Diagn Ther 2009;25:102-110

ENGRAFTMENT WINDOW: day 52-72

Sheep gestational age

Murine maternal and fetal viability

following in utero transplantation of

human HSC

Engraftment = marrow presence usually HSC phenotype

Expression = differentiation markers external to the marrow

Engraftment frequency or chimeric index

Pathobiology 1998; 66: 230-39

Reviews in Clinical and Experimental Hematology 8: 11-32, 1999

Day 54-65

Day 14-16

Engraftment frequency or chimeric index

Engraftment = marrow presence usually HSC phenotype

Expression = differentiation markers external to the marrow

Embryology of the thymus:

At a defined stage in development the

thymus demarcates and undergoes

vascularization with formation of the

medulla.

Gestational timing of demarcation:

Sheep beyond day 50

Mouse day 14-16

Immunology 1987; 62: 97–105.

Eur J Immunol 2000; 30:1948-1956 [PMID: 10940884]

World J Stem Cells 2013 April 26; 5(2): 43-52

doi:10.4252/wjsc.v5.i2.43

Human (xeno) and allogeneic grafts are

expandable after in utero transplantationGrowth Factor treatment

No

growth

factors

Hu

ma

n C

D4

5 +

ce

lls

in

tra

nsp

lan

ted

mic

e

Allogeneic in mice

• Prenatal tolerance induction and postnatal

nonmyeloablative bone marrow

transplantation

Blood. 2002; 100: 2225-2234

Xenogeneic (human) in sheep

• Postnatal administration of human

growth factors

Exp Hematol. 2007; 35: 1594–1600.

World J Stem Cells 2013 April 26; 5(2): 43-52

doi:10.4252/wjsc.v5.i2.43

Liver section obtained at 11 months post transplant from sheep transplanted with CB-derived CD34-Lin- cells and

stained with anti-human hepatocyte antibodyBlood First Edition online July 1, 2004; DOI 10.1182/blood-2004-01-0259

Tolerance to solid organ antigens/ Where are sheep NK cells?

Blood 2004; 104: 2582-2590

[PMID: 15231580 DOI: 10.1182/blood-2004-01-0259]

Stem Cell (SC) populations

studied for in vivo differentiative

capacity

1. Human fetal pancreas derived mesenchymal

SC

Exp Hematol 2010; 38: 311-320 [PMID:

20170708 DOI: 10.1016/j.exphem.2010.02.005]

2. Embryonic SC derived CD34 cells

• using mouse S17 BM stromal cell line and

• embryoid body formation and differentiation

in vitro

Exp Hematol 2010; 38: 516-525.e4 [PMID: 20227460

DOI: 10.1016/j.exphem.2010.03.002]

hfpSC (human fetal pancreatic SC) phenotype

in comparison to input population

hfpSC + for mesenchymal markers:

CD29, 44, 73,90 and 105

Real time quantitative PCR detection

of human DNA following

transplantation with hfpSC (human

fetal pancreatic stem cells)

• Four 1 mm3 samples from the pancreatic tail

• Limited sampling method

• Chimeric incidence: 79%

Circulating human insulin

in sheep 7 months to 2 years after

transplantation of hfpSC in utero

Exp Hematol 2010; 38: 311-320 [PMID: 20170708 DOI:

10.1016/j.exphem.2010.02.005]

Human C-peptide levels in sheep ~

2-3 years after transplantation of

human ESD-CD34+ SC during the

fetal tolerance window

A.D. Goodrich et al. Exp Hematol 2010; 38: 516-525.e4 [PMID:

20227460 DOI: 10.1016/j.exphem.2010.03.002]

In situ human islet activity in 2 sheep

transplanted with human stem cells

Chromogranin A is predominantly in α-cells

A.D. Goodrich et al. Exp Hematol 2010; 38: 516-525.e4 [PMID: 20227460 DOI: 10.1016/j.exphem.2010.03.002]

Summary and conclusion

1. Transplantation of allogeneic or xenogeneic

SCs during the fetal tolerance window in any

experimental animal renders that animal

tolerant to donor specific SCs and their

differentiated progeny including species

specific proteins

2. Any mulitpotent self-renewing SC

population would likely be effective

3. The (self-)tolerant environment allows

unfettered differentiation into both

hematopoietic and solid organ lineages

4. Present evidence suggests these grafts are

expandable

Summary and conclusion

continued

5. In utero transplantation is a powerful but

poorly understood investigative method that:

• should provide detailed understanding of

mechanisms underlying self-tolerance

• may provide alternative methods for the

investigation of biologic systems

• may allow alternative solutions to a number

of clinical problems

Acknowledgements

Alireza Torabi, M.D., Ph.D.

Jessica Reed, DVM, Ph.D.

Adel Ersek, Ph.D.

A Daisy Goodrich, Ph.D.

Esmail D Zanjani, Ph.D.

My Family: Janice, Aaron and Jedediah